Sc 67824

HeLa or U2OS cells were fixed with ice-cold methanol for 30 minutes at −20°C, and immunofluorescence microscopy was performed as described previously (O'Regan and Fry, 2009 (link)). Primary antibodies were against rootletin (1∶200, this study; or 1∶100, Santa Cruz, sc-67824), C-Nap1 (1∶1000, Fry et al., 1998a (link)), Nek2 (1∶200, BD Transduction Labs, 610594), GFP (0.5 µg/ml, Abcam, 6556), Cep135 (1∶1000, Kim et al., 2008 (link)), Myc (1∶1000, Cell Signaling Technology, 2276), γ-tubulin (1∶1000, Sigma T6557), cyclin B1 (0.2 µg/ml, Abcam, 2949) and pC-Nap1 AQDL and LLEK (0.5 µg/ml, this study). Secondary antibodies used were Alexa-Fluor-488-conjugated and Alexa-Fluor-594-conjugated goat anti-mouse-IgG and goat anti-rabbit-IgG (10 µg/ml, Invitrogen A11001 and A11012, respectively) or Alexa-Fluor-488-conjugated donkey anti-goat-IgG and Rhodamine-conjugated donkey anti-rabbit-IgG (5 µg/ml, Jackson 705-545-003 and 711-025-152, respectively). Images were captured using Volocity software (Improvision) on an inverted Nikon TE300 or Olympus BX51 microscope with a 100× oil objective. Intensity measurements were quantified using Volocity or ImageJ (v1.4.1) software and images were processed in Adobe Photoshop 4.0.

Antibodies were obtained from the indicated suppliers: goat anti-Rootletin for immunostaining (sc-67824) and mouse anti-Rootletin for immunoblotting (sc-374056; Santa Cruz Biotechnology, CA); mouse anti-acetylated tubulin (6–11B-1), mouse anti-FLAG (M2), and mouse anti-gamma-tubulin (T3559; Sigma-Aldrich, St. Louis, MO); mouse anti-Myc (9B11; Cell Signaling Technology, Danvers, MA); and rabbit anti-Pericentrin 2 (ab4448) and HRP-conjugated anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH, ab9385; Abcam, MA). Species-specific secondary antibodies were conjugated to Alexa Fluor 488 or 555 (Life Technologies, Grand Island, NY). Western blot analyses were performed as described previously31 (link).